Nano-theranostics for parkinson&#39;s disease

ABSTRACT

Provided are aggregate alpha-synuclein specific antibodies as well as fragments, derivatives, and variants thereof as well as method related thereto for the early diagnostic and treatment of Parkinson&#39;s Disease and other Lewy body- and Lewy neurite-based diseases. Assays, kits, systems, and nanoparticle encapsulated compositions related to the antibodies or fragments, derivatives, and variants thereof are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to provisionalapplication Ser. No. 62/804,805, filed Feb. 13, 2019, hereinincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 11, 2020, isnamed 2020-02-13_P12741US01_04793_Narasimhan_seq_ST25.txt and is 13,745bytes in size.

FIELD OF THE INVENTION

The present invention relates to an early screening test for Parkinson'sDisease and other Lewy body- and Lewy neurite-based diseases and relateddiseases and further used in treatment of such related diseases. Thepresent invention also relates to binding molecules, and methods ofproducing such binding molecules, that preferentially bind aggregatedalpha-synuclein without binding monomer alpha-synuclein.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) is a debilitating neurodegenerative disorder.The degenerative process is reportedly influenced by both environmentaland genetic factors. PD is multifactorial in origin, with aggregatedalpha-synuclein (αSyn) playing a major role in the pathologicaldevelopment and progression of PD. Alpha-synuclein as a monomer plays animportant role in neuronal homeostasis. Aggregated αSyn are constituentsof Lewy bodies (LBs) and Lewy neurites comprising primarily αSyndeposited in an aggregated amyloid fibril state are neuropathologicalhallmarks of PD, LB dementia, and other synucleoinpathies.

While LBs contain αSyn aggerates, the αSyn aggregates share many of thesame epitopes for binding molecules as the monomeric αSyn. This makesthem a poor target for development of novel management strategies,including diagnostics and therapeutics to stop, slow, or reverse PD. Bysharing the same epitopes as monomeric αSyn, binding molecules, such asantibodies, which recognize both are not useful as a diagnostic becausethey would detect both forms of αSyn and give false positive results.The binding molecules would also not be useful as a treatment for LBcontaining diseases because they would bind to and interrupt the normalfunction of αSyn and be detrimental to the patient.

To date, current commercial monoclonal antibodies (MAbs) are notsingularly specific in that they bind with both the aggregated andmonomeric forms of αSyn due to their shared epitopes. Thus, there are noantibodies suitable to be used for either a detection assay or as atherapeutic as the cross reactivity to both forms would assay for thephysiological αSyn as well as the aggregates and prevent the normalfunction of αSyn in a subject if used as a therapeutic. Due to thislimitation, there is not currently an assay for early accurate diagnosisof PD, nor are there efficacious drugs to prevent, slow progression, orreverse disease.

Thus, there is a need to develop an assay for early and accuratediagnosis of PD and other Lewy body- and Lewy neurite-based diseases andto provide a treatment which may prevent or block the progression of thedisease and/or reverse an established disease.

BRIEF SUMMARY OF THE INVENTION

The present invention provides anti-αSyn antibodies (Abs), fragments,and methods of making and using the same for the detection of potentialtoxic buildup of αSyn and for the treatment of αSyn aggregates in asubject to use as an early and accurate diagnosis of PD and other Lewybody- and Lewy neurite-based diseases and as a treatment to prevent,slow progression, or reverse these diseases. Samples may be taken from asubject and then assayed with anti-αSyn Abs or fragments in order todetect the presence of the aggregates in the samples. Anti-αSyn Abs orfragments may also be used to treat a subject with αSyn aggregates byadministering to the subject an effective amount to prevent αSynaggregation.

Applicants have identified both polyclonal (PAbs) and monoclonal (MAbs)antibodies and fragments thereof having a high affinity specifically forthe toxic aggregates of αSyn. These antibodies and fragments show ahigher binding affinity for aggregate αSyn than to monomer αSyn. In someembodiments the antibodies or fragments bind specifically to aggregateαSyn and not to the physiological monomer of αSyn. In some embodiments,the antibody or fragment belongs to the IgG family. In otherembodiments, the antibody or fragment belongs to the IgM family. In someembodiments polyclonal Abs are made from injecting a different speciesαSyn into a subject and then isolating the resulting Abs from thatsubject. In some embodiments, the monoclonal Abs are made fromhybridomas. In further embodiments, the Abs made in a different speciesmay be modified in order to more safely administered to a subject, forexample humanized or chimeric Abs. In some embodiments, fragments of theAbs may also be used in place of Abs. In some embodiments are antibodiesthat bind to same epitopes as the disclosed antibodies. In yet furtherembodiments the antibodies are encoded by a polynucleotide.

The Abs or fragments may be used in any organism that produces αSyn andaggregates for detection, diagnostics, and treatment. In someembodiments, the antibody or fragment binds to the human αSyn. In otherembodiments, the antibody binds to animal αSyn aggregates and/ormonomers. In further embodiments, the antibody may bind to primate,rodent, canine, feline, ungulate, mustelid, lagomorph, chondrichthyes,or osteichthyes.

To enhance their use, the antibodies or fragments may be conjugated witha variety of compounds. In order to be used in a detection system, insome embodiments, the Abs or fragments are conjugated with a fluorophoreor enzyme that may be detected in a system. In other embodiments, asecondary antibody, which is bonded to a fluorophore or enzyme, may bindto the αSyn aggregate antibody or fragment in a detection system.

In other embodiments, the Abs or fragments may be loaded ontonanoparticles to enhance their effectiveness when used as a therapy. Thenanoparticles may allow the Abs or fragments to avoid degradation,uptake into the wrong organ, or for enhanced passage across the bloodbrain barrier.

In yet another embodiment, the Abs or fragments may be provided in a kitfor a detection system. The kit would include at least the antibodies orfragments for binding to αSyn aggregates in a sample and instructionsfor their use.

In an additional embodiment, the Abs or fragments may be used in asystem to detect aggregated αSyn in a sample.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed descriptions, which show and describeillustrative embodiments of the invention. Accordingly, the figures anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a blot using anti human αSyn aggregate polyclonal antibodygenerated in mice (HA-PAb) show that toxic aggregates of recombinanthuman αSyn include unique epitopes/determinants that are absent in thephysiological monomeric form of αSyn.

FIG. 2 shows that the HA-PAbs demonstrate exemplary discriminationbetween the toxic and physiological forms of αSyn compared tocommercially available monoclonal antibodies.

FIG. 3 shows detection of aggregated αSyn in cerebral spinal fluid andserum of Parkinson's disease (PD) patients when compared to age-matched(AM) controls.

FIG. 4 shows hybridoma generated monoclonal anti human αSyn aggregateantibody (HA-MAb) having a differential reactivity with serum samplesfrom PD patients and AM controls as determined by immunoblotting.

FIG. 5A shows a graphical representation of the IgM 3A8 HA-MAb used inan indirect competitive Enzyme Immuno Assay (EIA) can detect increasingamounts of competing αSyn with a high sensitivity and specificity. FIG.5B shows a graphical representation of the EIA resulting indiscrimination between PD patients and MA controls at differentconcentrations of sample.

FIG. 6 is a representative denaturing agarose gel with profiles of totalcellular RNA isolated from hybridoma clones 3A8 (lane 1) and 6G7 (lane2) and a molecular ladder (land M), confirming the presence of the 28Sand 18S bands for quality assessment.

FIG. 7 is a representative agarose gel showing the variable heavy chain(VH) and variable light (VL) chain domains encoding gene fragmentsamplified by Rapid Amplification of cDNA Ends (RACE) for 3A8 (3A8VH,lane 1, and 3A8VL, lane 2) and 6G7 (6G7VH, lane 3, and 6G7VL, land 4)next to a molecular ladder (lane M) having the expected size of about400 bp.

FIG. 8 is a graphical representation of the predicted 3D structure ofthe scFvs for 3A8 and 6G7.

DETAILED DESCRIPTION

Synucleinopathic diseases or synucleinopathies are a diverse group ofneurodegenerative disorders that share a common pathologic lesioncomposed of aggregates of insoluble α-synuclein (αSyn) protein inselectively vulnerable populations of neurons and glia. These disordersinclude Parkinson's disease (PD), Parkinson's Disease Dementia (PDD),dementia with Lewy bodies (DLB), juvenile-onset generalized neuroaxonaldystrophy (Hallervorden-Spatz disease), pure autonomic failure (PAF),multiple system atrophy (MSA) and neurodegeneration with brain ironaccumulation type-1 (NBIA-I). Clinically, they are characterized by achronic and progressive decline in motor, cognitive, behavioral, andautonomic functions, depending on the distribution of the lesions.

Parkinson's disease is an age-dependent neurodegenerative disease withunknown etiology. It is believed that sporadic Parkinson's diseaseresults from a combination of genetic vulnerability and environmentalinsults. It is further believed that Parkinson's disease (PD) whiletriggered by disparate mechanisms follows a shared pathophysiologicpathway. One shared node is the involvement of αSyn. Linkage of thisprotein with Parkinson's disease pathogenesis has been established bythe identification of both point mutations and triplication of the genein familial cases, the localization of αSyn to Lewy bodies, one of thehallmark pathological features of Parkinson's disease, and thecorrelation of αSyn expression and disease pathology in neurotoxicmodels of Parkinson's disease. Further evidence indicates thatparticular forms of αSyn (e.g., misfolded and αSyn bonded dopamine) areinvolved in sporadic disease.

Synucleins are small, soluble proteins expressed primarily in neuraltissue and in certain tumors. The family includes three known proteins:αSyn, βSyn, and γSyn. All synuclein have in common a highly conservedα-helical lipid-binding motif with similarity to the class-A2lipid-binding domains of the exchangeable apolipoproteins. Synucleinfamily members are not found outside vertebrates, although they havesome conserved structural similarity with plant late-embryo-abundantproteins. The α- and β-synuclein proteins are found primarily in braintissue, where they are seen mainly in presynaptic terminals. The γSynprotein is found primarily in the peripheral nervous system and retina,but its expression in breast tumors is a marker for tumor progression.Normal cellular functions have not been determined for any of thesynuclein proteins, although some data suggest a role in the regulationof membrane stability and/or turnover. Mutations in αSyn are associatedwith rare familial cases of early-onset Parkinson's disease, and theprotein accumulates abnormally in Parkinson's disease, Alzheimer'sdisease, and several other neurodegenerative illnesses. For review see,e.g., George, Genome Biol. 3 (2002), reviews3002.1-reviews3002.6published online Dec. 20, 2001, in which Table 1 catalogs the uniquemembers of the synuclein family that are currently listed in GenBank,the disclosure content of which is incorporated herein by reference.

Alpha-synuclein was originally identified in human brains as theprecursor protein of the nonβ-amyloid component of (NAC) of Alzheimer'sdisease (AD) plaques. Alpha-synuclein, also termed the precursor of thenon-Aβ component of AD amyloid (NACP), is a protein of 140 amino acids.Alpha-synuclein exists in its native form as a random coil; however,changes in pH, molecular crowding, heavy metal content, and dopaminelevels all affect protein conformation. Changes in conformation tooligomeric, proto-fibrillar, fibrillar, and aggregate moieties arethought to regulate protein toxicity. Increasing evidence indicates thatdopamine-adducted αSyn has a faster time course to fibril formationcompared to non-adducted protein. Furthermore, dopamine in thebackground of αSyn overexpression is toxic.

The embodiments of this invention are not limited to particular methodsof selection, methods of production, and compositions, which can varyand may be understood by skilled artisans. It is further to beunderstood that all terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting in any manner or scope. For example, as used in thisspecification and the appended claims, the singular forms “a,” “an” and“the” can include plural referents unless the content clearly indicatesotherwise. Further, all units, prefixes, and symbols may be denoted inits SI accepted form.

Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange. Throughout this disclosure, various aspects of this invention arepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges, fractions,and individual numerical values within that range. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 3, 4, 5, and 6,and decimals and fractions, for example, 1.2, 3.8, 1½, and 4¾ Thisapplies regardless of the breadth of the range.

The phrase “and/or,” when used between elements in a list, is intendedto mean either (1) that only a single listed element is present, or (2)that more than one element of the list is present. For example, “A, B,and/or C” indicates that the selection may be A alone; B alone; C alone;A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may beused interchangeably with “at least one of” or “one or more of” theelements in a list.

Definitions

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided. Units, prefixes, and symbols maybe denoted in their SI accepted form. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Numeric ranges are inclusive of the numbersdefining the range and include each integer within the defined range.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes. Theterms defined below are more fully defined by reference to thespecification as a whole.

As used herein, the terms “α-synuclein”, “alpha-synuclein”, “αSyn” and“αSyn” are used interchangeable to specifically refer to the nativemonomer form of .alpha.-synuclein. The term “αSyn” is also used togenerally identify other conformers of αSyn, for example, αSyn bonded todopamine-quinone (DAQ) and oligomers or aggregates of αSyn. The term“αSyn” is also used to refer collectively to all types and forms ofαSyn.

The protein sequence for human αSyn isMDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 1). The amino acid sequenceof αSyn can be retrieved from the literature and pertinent databases;see e.g. GenBank, assession number NP_000336, Swissprot: locus SYUAHUMAN, accession number P37840. The non-Aβ component of AD amyloid (NAC)is derived from αSyn. NAC, a highly hydrophobic domain within αSyn, is apeptide consisting of at least 28 amino acids residues (residues 60-87)and optionally 35 amino acid residues (residues 61-95). NAC displays atendency to form a beta-sheet structure. The amino acid sequences of NACare described in Jensen et al., Biochem. J. 310 (1995), 91-94; GenBankaccession number S56746 and Ueda et al., PNAS USA 90 (1993), 1282-11286.

As used herein, “disaggregated αSyn” or fragments thereof, includingNAC, means monomeric peptide units. Disaggregated αSyn or fragmentsthereof are generally soluble and are capable of self-aggregating toform soluble oligomers. Oligomers of αSyn and fragments thereof areusually soluble and exist predominantly as α-helices. Monomeric αSyn maybe prepared in vitro by dissolving lyophilized peptide in neat DMSO withsonication. The resulting solution is centrifuged to remove anyinsoluble particulates.

As used herein, “aggregated αSyn” or fragments thereof, including NAC,means oligomers of αSyn or fragments thereof which have associate intoinsoluble β-sheet assemblies. Aggregated αSyn or fragments thereof,including NAC, means also means fibrillar polymers. Fibrils are usuallyinsoluble. In some embodiments, antibodies bind either soluble αSyn orfragments thereof or aggregated αSyn or fragments thereof. In otherembodiments, antibodies bind to oligomers of αSyn more strongly than tomonomeric forms or fibrillar forms. In yet additional embodiments,antibodies bind both soluble and aggregated αSyn or fragments thereof,and optionally oligomeric forms as well.

The human anti-αSyn antibodies disclosed herein specifically bind αSynand epitopes thereof and to various conformations of αSyn and epitopesthereof. For example, disclosed herein are antibodies that specificallybind αSyn, αSyn in its native monomer form, full-length and truncatedαSyn and αSyn aggregates. As used herein, reference to an antibody that“specifically binds”, “selectively binds”, or “preferentially binds”αSyn refers to an antibody that does not bind other unrelated proteins.In one example, an αSyn antibody disclosed herein can bind αSyn or anepitope thereof and show no binding above about 1.25, about 1.5, about1.75, or about 2 times background for other proteins. An antibody that“specifically binds” or “selectively binds” αSyn conformer refers to anantibody that does not bind all conformations of αSyn, i.e., does notbind at least one other αSyn conformer. For example, disclosed hereinare antibodies that can distinguish among monomeric and aggregated formsof αSyn, human and mouse αSyn; full-length αSyn and truncated forms aswell as human αSyn versus β- and γ-synuclein. Since the human anti-αSynantibodies of the present invention have been isolated from a pool ofelderly subjects with no signs of Parkinsonism and exhibiting anαSyn-specific immune response the anti-αSyn antibodies of the presentinvention may also be called “human auto-antibodies” in order toemphasize that those antibodies were indeed expressed by the subjectsand have not been isolated from, for example a human immunoglobulinexpressing phage library, which hitherto represented one common methodfor trying to provide human-like antibodies.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,methylation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids. A polypeptide may be derived from a natural biological source orproduced by recombinant technology but is not necessarily translatedfrom a designated nucleic acid sequence. It may be generated in anymanner, including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to antibodies or antibody polypeptides of thepresent invention include any polypeptides which retain at least some ofthe antigen-binding properties of the corresponding native bindingmolecule, antibody, or polypeptide. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of antibodies and antibody polypeptides ofthe present invention include fragments as described above, and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants may occur naturally orbe non-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of αSyn specific binding molecules,e.g., antibodies and antibody polypeptides of the present invention, arepolypeptides which have been altered so as to exhibit additionalfeatures not found on the native polypeptide. Examples include fusionproteins. Variant polypeptides may also be referred to herein as“polypeptide analogs”. As used herein a “derivative” of a bindingmolecule or fragment thereof, an antibody, or an antibody polypeptiderefers to a subject polypeptide having a residue chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides which contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” or “operablylinked” if induction of promoter function results in the transcriptionof mRNA encoding the desired gene product and if the nature of thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operably associatedwith a nucleic acid encoding a polypeptide if the promoter was capableof effecting transcription of that nucleic acid. The promoter may be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. Other transcription control elements,besides a promoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein.

As used herein, a “binding molecule” relates primarily to antibodies,and fragments thereof, but may also refer to other non-antibodymolecules that bind to .alpha.-synuclein including but not limited tohormones, receptors, ligands, major histocompatibility complex (MHC)molecules, chaperones such as heat shock proteins (HSPs) as well ascell-cell adhesion molecules such as members of the cadherin, intergrin,C-type lectin and immunoglobulin (Ig) superfamilies. Thus, for the sakeof clarity only and without restricting the scope of the presentinvention most of the following embodiments are discussed with respectto antibodies and antibody-like molecules which represent the preferredbinding molecules for the development of therapeutic and diagnosticagents.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is an αSyn-binding molecule whichcomprises at least the variable domain of a heavy chain, and normallycomprises at least the variable domains of a heavy chain and a lightchain. Basic immunoglobulin structures in vertebrate systems arerelatively well understood; see, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or,epsilon, (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4).It is the nature of this chain that determines the “class” of theantibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulinsubclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules as merely an example. For example,with regard to IgG, a standard immunoglobulin molecule comprises twoidentical light chain polypeptides of molecular weight approximately23,000 Daltons, and two identical heavy chain polypeptides of molecularweight 53,000-70,000. The four chains are typically joined by disulfidebonds in a “Y” configuration wherein the light chains bracket the heavychains starting at the mouth of the “Y” and continuing through thevariable region. IgM share the similar Y structure, however theygenerally resemble a pentamer or hexamer, depending on the organism oforigin, of IgG joined in various ways through disulfide bonds.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three-dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the V_(H) and V_(L) chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to αSyn is denoted herein interchangeably as a“binding fragment” or an “immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three-dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a β-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of the β-sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196 (1987), 901-917, which are incorporated herein by referencein their entireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (CDR) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are bothincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE 1 Numbering of all CDR definitions in Table 1 is according to thenumbering conventions set for by Kabat et al. CDR Definitions KabatChothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-28 VH CDR3  95-102  95-102VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously-assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system.

Antibodies or antigen-binding fragments, immunospecific fragments,variants, fusion proteins, or derivatives thereof of the inventioninclude, but are not limited to, polyclonal, monoclonal, multispecific,human, humanized, primatized, murinized or chimeric antibodies, singlechain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFvs), single-chain antibodies,disulfide-linked Fvs (sdFv), fragments comprising either a V_(L) orV_(H) domain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies disclosed herein). ScFv molecules are known in the art andare described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin orantibody molecules of the invention can be of any type (e.g., IgG, IgE,IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass of immunoglobulin molecule.

In a particularly preferred embodiment, the antibody of the presentinvention is not a polyclonal antibody, i.e. it substantially consistsof one particular antibody species rather than being a mixture obtainedfroth a plasma immunoglobulin sample.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are αSyn-binding fragments also comprising anycombination of variable region(s) with a hinge region, CH1, CH2, and CH3domains. Antibodies or immunospecific fragments thereof of the presentinvention may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks).

In one aspect, the antibody of the present invention is a humanmonoclonal antibody isolated from a human. Optionally, the frameworkregion of the human antibody is aligned and adopted in accordance withthe pertinent human germ line variable region sequences in the database;see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). For example, amino acidsconsidered to potentially deviate from the true germ line sequence couldbe due to the PCR primer sequences incorporated during the cloningprocess. Compared to artificially generated human-like antibodies suchas single chain antibody fragments (scFvs) from a phage displayedantibody library or xenogeneic mice the human monoclonal antibody of thepresent invention is characterized by (i) being obtained using the humanimmune response rather than that of animal surrogates, i.e. the antibodyhas been generated in response to natural .alpha.-synuclein in itsrelevant conformation in the human body, (ii) having protected theindividual or is at least significant for the presence of.alpha.-synuclein, and (iii) since the antibody is of human origin therisks of cross-reactivity against self-antigens is minimized. Thus, inaccordance with the present invention the terms “human monoclonalantibody”, “human monoclonal autoantibody”, “human antibody” and thelike are used to denote an αSyn binding molecule which is of humanorigin, i.e. which has been isolated from a human cell such as a B cellor hybridoma thereof or the cDNA of which has been directly cloned frommRNA of a human cell, for example a human memory B cell. A humanantibody is still “human” even if amino acid substitutions are made inthe antibody, e.g., to improve binding characteristics.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al., are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

In other aspects of the invention, an antibody may be raised in a firstorganism then be modified to be more similar to a different organism,such as a murinized or humanized antibody. For example, and as usedherein, the term “murinized antibody” or “murinized immunoglobulin”refers to an antibody comprising one or more CDRs from a human antibodyof the present invention; and a human framework region that containsamino acid substitutions and/or deletions and/or insertions that arebased on a mouse antibody sequence. The human immunoglobulin providingthe CDRs is called the “parent” or “acceptor” and the mouse antibodyproviding the framework changes is called the “donor”. Constant regionsneed not be present, but if they are, they are usually substantiallyidentical to mouse antibody constant regions, i.e. at least about85-90%, preferably about 95% or more identical. Hence, in someembodiments, a full length murinized human heavy or light chainimmunoglobulin contains a mouse constant region, human CDRs, and asubstantially human framework that has a number of “murinizing” aminoacid substitutions. Typically, a “murinized antibody” is an antibodycomprising a murinized variable light chain and/or a murinized variableheavy chain. For example, a murinized antibody would not encompass atypical chimeric antibody, e.g., because the entire variable region of achimeric antibody is non-mouse. A modified antibody that has been“murinized” by the process of “murinization” binds to the same antigenas the parent antibody that provides the CDRs and is usually lessimmunogenic in mice, as compared to the parent antibody. Conversely, a“humanized antibody” is an antibody comprising a humanized variablelight change and/or a humanized variable heavy chain, with asubstantially murine framework.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for in vivo therapeutic and diagnosticapplications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, a peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofa Syn.

By “specifically binding”, or “specifically recognizing”, usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen-binding domain,and that the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D”.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to hind a αSyn or afragment or variant thereof with an off rate (k(off)) of less than orequal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, or 10⁻³ sec⁻¹. Morepreferably, an antibody of the invention may be said to bind αSyn or afragment or variant thereof with an off rate (k(off)) less than or equalto 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹,10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind α-synucleinor a fragment or variant thereof with an on rate (k(on)) of greater thanor equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹sec⁻¹. More preferably, an antibody of the invention may be said to bindα-synuclein or a fragment or variant thereof with an on rate (k(on))greater than or equal to 10⁵ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valencies of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N.Y.(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC₅₀, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross-reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to αSyn. Preferredbinding affinities include those with a dissociation constant or K_(d)less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁶M 5×10⁻⁷ M, 10⁻⁷ M 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

As previously indicated, the subunit structures and three-dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the V_(H) domain and is amino terminal to the hinge regionof an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit). The CH2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system). InIgM molecules, the various heavy chains making up different subunits usedisulfide bonds to the other heavy chains to form an inner ringsurrounded by the Y shape epitope binding regions.

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components; by whatever means including chemical conjugationor recombinant means. Art “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one aspect, a sample can compriseblood or serum, cerebrospinal fluid (“CSF”), or urine. In other aspects,a sample can comprise whole blood, plasma, B cells enriched from bloodsamples, and cultured cells (e.g., B cells from a subject). A sample canalso include a biopsy or tissue sample including neural tissue. In stillother aspects, a sample can comprise whole cells and/or a lysate of thecells. Blood samples can be collected by methods known in the art.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development ofParkinsonism. Beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the manifestation of the condition or disorder is to beprevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

The term “sufficient amount of time,” as used herein, refers to the timeit takes for a compound, material, composition comprising a compound ofthe present invention, or an organism which is effective for producingsome desired effect in at least a sub-population of cells.

As used herein, “substantially free” may refer to any component that thecomposition of the invention lacks or mostly lacks. When referring to“substantially free” it is intended that the component is notintentionally added to compositions of the invention. Use of the term‘substantially free” of a component allows for trace amounts of thatcomponent to be included in compositions of the invention because theyare present in another component. However, it is recognized that onlytrace or de minimus amounts of a component will be allowed when thecompositions is said to be “substantially free” of that component.Moreover, the term if a composition is said to be “substantially free”of a component, if the component is present in trace or de minimusamounts it is understood that it will not affect the effectiveness ofthe compositions. It is understood that if an ingredient is notexpressly included herein or its possible inclusion is not statedherein, the invention composition may be substantially free of thatingredient. Likewise, the express inclusion of an ingredient allows forits express exclusion thereby allowing a composition to be substantiallyfree of that expressly stated ingredient.

Antibodies

The present invention generally relates to human anti-αSyn antibodiesand antigen-binding fragments thereof, which preferably demonstrate theimmunological binding characteristics and/or biological properties asoutlined for the antibodies illustrated in the Examples.

In one embodiment, the present invention is directed to an anti-αSynantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody specifically binds to the same epitope of αSyn as thereference antibodies illustrated in the Examples. As illustrated in theExamples, the various antibodies from either the polyclonal ormonoclonal groups bind to aggregates of αSyn but not to the physiologic,monomeric form of αSyn. For the polyclonal antibodies this was achievedthrough the removal of antibodies that bind to epitopes found on themonomer αSyn or to epitopes found in other synucleins. Selection can bedone using any method known in the art, such as, but not limited to,column purification. For the monoclonal antibodies, the murine spellcells were fused with myelomas to form hybridomas. The hybridomas werethen assayed for specific binding to aggregated αSyn and not havingcross-binding to monomeric αSyn.

In one embodiment, the antibody of the present invention exhibits thebinding properties of the exemplary antibodies as described in any oneof the Examples.

The present invention further exemplifies several such bindingmolecules, e.g. antibodies and binding fragments thereof which may becharacterized by comprising in their variable region, e.g. bindingdomain at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) variable region comprising any one of the amino acidsequences of the antibodies illustrated in the Examples. However, asdiscussed in the following the person skilled in the art is well awareof the fact that in addition or alternatively CDRs may be used, whichdiffer in their amino acid sequence by one, two, three or even moreamino acids in case of CDR2 and CDR3.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the V_(H) and/orV_(L) of the antibodies illustrated in the Examples. Alternatively, theantibody of the present invention is an antibody or antigen-bindingfragment, derivative or variant thereof, which competes for binding toαSyn with at least one of the antibodies having the V_(H) and/or V_(L)region as illustrated in the Examples. Those antibodies may be humanizedmurine or human-murine chimeric antibodies, in particular fortherapeutic applications. Alternatively, the antibody is a murine,murinized and chimeric murine-human antibody, which are particularlyuseful for diagnostic methods and studies in animals.

As mentioned above, due to its specificity the polyclonal and monoclonalantibodies of the present invention will recognize epitopes which are ofparticular physiological relevance and show a specificity not found incommercially available antibodies. Accordingly, it is prudent tostipulate that the epitope of the human anti-αSyn antibody of thepresent invention is unique and no other antibody which is capable ofbinding to the epitope recognized by the polyclonal or monoclonalantibody of the present invention exists. Therefore, the presentinvention also extends generally to anti-αSyn antibodies and αSynbinding molecules which compete with the polyclonal and monoclonalantibody of the present invention for specific binding to αSyn. Thepresent invention is more specifically directed to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody specifically binds to the same epitope of αSyn as a referenceantibody as illustrated in the Examples.

Competition between antibodies may be determined, for examples, by anassay in which the immunoglobulin under test inhibits specific bindingof a reference antibody to a common antigen, such as αSyn. Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (ETA), sandwich competition assay, enzymeimmuno assay (EIA); see Stahli et al., Methods in Enzymology 9 (1983),242-253; solid phase direct biotin-avidin EIA; see Kirkland et al., J.Immunol. 137 (1986), 3614-3619 and Cheung et al., Virology 176 (1990),546-552; solid phase direct labeled assay, solid phase direct labeledsandwich assay; see Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Press (1988); solid phase direct label RIA using I¹²⁵label; see Morel et al, Molec. Immunol. 25 (1988), 7-15 and Moldenhaueret al., Scand. J. Immunol. 32 (1990), 77-82. Typically, such an assayinvolves the use of purified αSyn or aggregates thereof bound to a solidsurface or cells bearing either of these, an unlabelled testimmunoglobulin and a labeled reference immunoglobulin, i.e. themonoclonal antibody of the present invention. Competitive inhibition ismeasured by determining the amount of label bound to the solid surfaceor cells in the presence of the test immunoglobulin. Usually the testimmunoglobulin is present in excess. Antibodies identified bycompetition assay (competing antibodies) include antibodies binding tothe same epitope as the reference antibody and antibodies binding to anadjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur. Usually, when acompeting antibody is present in excess, it will inhibit specificbinding of a reference antibody to a common antigen by at least 50% or75%. Hence, the present invention is further drawn to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody competitively inhibits a reference antibody selected from theantibodies illustrated in the Examples from binding to αSyn.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of or consisting of animmunoglobulin heavy chain variable region (V_(H)) or light chainvariable region (V_(L), together V), where at least one of V-CDRs of theheavy or light chain variable region or at least two of the V-CDRs ofthe heavy or light chain variable region are at least 80%, 85%, 90% or95% identical to reference heavy or light chain V-CDR1, V-CDR2 or V-CDR3amino acid sequences from the antibodies disclosed herein.Alternatively, the V-CDR1, V-CDR2 and V-CDR3 regions of the V are atleast 80%, 85%, 90% or 95% identical to reference heavy chain V-CDR1,V-CDR2 and V-CDR3 amino acid sequences from the antibodies disclosedherein.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin variable region (V) of the light and/or heavy chain inwhich the V-CDR1, V-CDR2 and V-CDR3 regions have polypeptide sequenceswhich are identical to the V-CDR1, V-CDR2 and V-CDR3 groups of theantibodies disclosed herein.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin variable region (V) of the heavy or light chain in whichthe V-CDR1, V-CDR2 and V-CDR3 regions have polypeptide sequences whichare identical to the V-CDR1, V-CDR2 and V-CDR3 groups of the antibodiesdisclosed herein, except for one, two, three, four, five, or six aminoacid substitutions in any one V-CDR. In certain embodiments the aminoacid substitutions are conservative.

An immunoglobulin or its encoding cDNA may be further modified. Thus, ina further embodiment the method of the present invention comprises anyone of the step(s) of producing, for example, a chimeric antibody,murinized or humanized antibody, single-chain antibody, Fab-fragment,bispecific antibody, fusion antibody, labeled antibody or an analog ofany one of those. Corresponding methods are known to the person skilledin the art and are described, e.g., in Harlow and Lane “Antibodies, ALaboratory Manual”, CSH Press, Cold Spring Harbor (1988). Whenderivatives of said antibodies are obtained by the phage displaytechnique, surface plasmon resonance as employed in the BIAcore systemcan be used to increase the efficiency of phage antibodies which bind tothe same epitope as that of any one of the antibodies described herein(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13). The production of chimericantibodies is described, for example, in international applicationWO89/09622. Methods for the production of humanized antibodies aredescribed in, e.g., European application EP-A1 0 239 400 andinternational application WO90/07861. A further source of antibodies tobe utilized in accordance with the present invention are so-calledxenogeneic antibodies. The general principle for the production ofxenogeneic antibodies such as human-like antibodies in mice is describedin, e.g., international applications WO91/10741, WO94/02602, WO96/34096and WO 96/33735. As discussed above, the antibody of the invention mayexist in a variety of forms besides complete antibodies; including, forexample, Fv, Fab and F(ab)₂, as well as in single chains, for examplescFv; see e.g. international application WO88/09344.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international applicationWO00/30680 for corresponding technical details.

Additionally, the present invention encompasses peptides including thosecontaining a binding molecule as described above, for example containingthe CDR3 region of the variable region of any one of the mentionedantibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment thereof which is oriented towards the humananti-αSyn antibodies of the present invention and display the mentionedproperties, i.e. which specifically recognize aggregate αSyn. Suchantibodies and binding molecules can be tested for their bindingspecificity and affinity by methods known in the art such as, but notlimited to, ELISA and Western Blot and immunohistochemistry.Furthermore, preliminary results of subsequent experiments performed inaccordance with the present invention revealed that the human anti-αSynantibody of the present invention recognizes αSyn inclusion bodiespresent on human brain sections of patients who suffered from dementiawith Lewy bodies (DLB) or Parkinson's disease (PD). Thus, in aparticular preferred embodiment of the present invention, the humanantibody or binding fragment, derivative or variant thereof recognizesαSyn on human DLB or PD brain sections.

As an alternative to obtaining immunoglobulins directly from serum, theculture of immortalized B cells, B memory cells, or hybridomas, theimmortalized cells can be used as a source of rearranged heavy chain andlight chain loci for subsequent expression and/or genetic manipulation.Rearranged antibody genes can be reverse transcribed from appropriatemRNAs to produce cDNA. If desired, the heavy chain constant region canbe exchanged for that of a different isotype or eliminated altogether.The variable regions can be linked to encode single chain Fv regions.Multiple Fv regions can be linked to confer binding ability to more thanone target or chimeric heavy and light chain combinations can beemployed. Once the genetic material is available, design of analogs asdescribed above which retain both their ability to bind the desiredtarget is straightforward. Methods for the cloning of antibody variableregions and generation of recombinant antibodies are known to the personskilled in the art and are described, for example, Gilliland et al.,Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997),1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, for example mammalianor bacterial cells. Typical mammalian cell lines useful for this purposeinclude, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are preferably designed to include signalpeptides so that the resulting antibodies are secreted into the medium;however, intracellular production is also possible.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antibody or equivalent binding molecule ofthe present invention, in case of the antibody preferably at least avariable region of an immunoglobulin chain of the antibody describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) of the variable region of the said antibody.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988),323-327. Thus, the present invention also relates to antibodies whereinone or more of the mentioned CDRs comprise one or more, preferably notmore than two amino acid substitutions. Preferably, the antibody of theinvention comprises in one or both of its immunoglobulin chains two orall three CDRs of the variable regions of the antibodies illustrated inthe Examples.

Binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention, as known by those ofordinary skill in the art, can comprise a constant region which mediatesone or more effector functions. For example, binding of the C1 componentof complement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the present invention include anantibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of αSyn aggregation and deposition, reduced serum half-life, orincreased serum half-life when compared with a whole, unaltered antibodyof approximately the same immunogenicity. For example, certainantibodies for use in the diagnostic and treatment methods describedherein are domain deleted antibodies which comprise a polypeptide chainsimilar to an immunoglobulin heavy chain, but which lack at least aportion of one or more heavy chain domains. For instance, in certainantibodies, one entire domain of the constant region of the modifiedantibody will be deleted, for example, all or part of the CH2 domainwill be deleted. In other embodiments, certain antibodies for use in thediagnostic and treatment methods described herein have a constantregion, e.g., an IgG heavy chain constant region, which is altered toeliminate glycosylation, referred to elsewhere herein as aglycosylatedor “agly” antibodies. Such “agly” antibodies may be preparedenzymatically as well as by engineering the consensus glycosylationsite(s) in the constant region. While not being bound by theory, it isbelieved that “agly” antibodies may have an improved safety andstability profile in vivo. Methods of producing aglycosylatedantibodies, having desired effector function are found for example ininternational application WO2005/018572, which is incorporated byreference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing αSyn localization. Inother cases, it may be that constant region modifications consistentwith the instant invention moderate complement binding and thus reducethe serum half-life and nonspecific association of a conjugatedcytotoxin. Yet other modifications of the constant region may be used tomodify disulfide linkages or oligosaccharide moieties that allow forenhanced localization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as αSynlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences to increase the cellularuptake of antibodies by way of example by enhancing receptor-mediatedendocytosis of antibodies via Fcγ receptors, Fcμ receptors, LRP, or Thy1receptors or by ‘SuperAntibody Technology’, which is said to enableantibodies to be shuttled into living cells without harming them (ExpertOpin. Biol. Ther. (2005), 237-241). For example, the generation offusion proteins of the antibody binding region and the cognate proteinligands of cell surface receptors or bi- or multi-specific antibodieswith specific sequences biding to αSyn as well as a cell surfacereceptor may be engineered using techniques known in the art. In certainantibodies, or antigen-binding fragments, variants, or derivativesthereof described herein, the Fc portion may be mutated or exchanged foralternative protein sequences or the antibody may be chemically modifiedto increase its blood brain barrier penetration.

Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be made or manufactured using techniques that are known inthe art. In certain embodiments, antibody molecules or fragments thereofare “recombinantly produced,” i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibody molecules orfragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In particular preferred embodiments, antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., internationalapplications WO98/52976 and WO00/34317. For example, V_(H) and V_(L)sequences from the starting antibody are analyzed and a human T cellepitope “map” from each V region showing the location of epitopes inrelation to complementarity determining regions (CDRs) and other keyresidues within the sequence. Individual T cell epitopes from the T cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative V_(H) and V_(L) sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of binding polypeptides,e.g., αSyn-specific antibodies or immunospecific fragments thereof foruse in the diagnostic and treatment methods disclosed herein, which arethen tested for function. Typically, between 12 and 24 variantantibodies are generated and tested. Complete heavy and light chaingenes comprising modified V and human C regions are then cloned intoexpression vectors and the subsequent plasmids introduced into celllines for the production of whole antibody. The antibodies are thencompared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), said references incorporatedby reference in their entireties. The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. In certain embodiments, antibodies of the presentinvention are derived from human B cells which have been immortalizedvia transformation with Epstein-Barr virus, as described herein.

In the well-known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., spleen cells derived from a mouse, are fused with an immortaltumor cell line (e.g., a myeloma cell line), thus, producing hybridcells or “hybridomas” which are both immortal and capable of producingthe genetically coded antibody of the B cell. The resulting hybrids aresegregated into single genetic strains by selection, dilution, andre-growth with each individual strain comprising specific genes for theformation of a single antibody. They produce antibodies, which arehomogeneous against a desired antigen and, in reference to their puregenetic parentage, are termed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). After hybridoma cells are identified thatproduce antibodies of the desired specificity, affinity and/or activity,the clones may be subcloned by limiting dilution procedures and grown bystandard methods; see, e.g., Goding, Monoclonal Antibodies: Principlesand Practice, Academic Press, pp 59-103 (1986). It will further beappreciated that the monoclonal antibodies secreted by the subclones maybe separated from culture medium, ascites fluid or serum by conventionalpurification procedures such as, for example, protein-A, hydroxylapatitechromatography, gel electrophoresis, dialysis or affinitychromatography.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific immunoglobins, such as IgGs orIgMs, that meet the screening criteria. Cells from positive wells can beisolated. Individual Ig-producing B cells can be isolated by FACS or byidentifying them in a complement-mediated hemolytic plaque assay.Ig-producing B cells can be micromanipulated into a tube and the V_(H)and V_(L) genes can be amplified using, e.g., RT-PCR. The V_(H) andV_(L) genes can be cloned into an antibody expression vector andtransfected into cells (e.g., eukaryotic or prokaryotic cells) forexpression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain. Such fragments are sufficient for use, for example, inimmunodiagnostic procedures involving coupling the immunospecificportions of immunoglobulins to detecting reagents such as radioisotopes.

In one embodiment, an antibody of the invention comprises at least oneheavy or light chain CDR of an antibody molecule. In another embodiment,an antibody of the invention comprises at least two CDRs from one ormore antibody molecules. In another embodiment, an antibody of theinvention comprises at least three CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least four CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least five CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least six CDRs from one or more antibodymolecules. Exemplary antibody molecules comprising at least one CDR thatcan be included in the subject antibodies are described herein.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably by recombinant expression techniques asdescribed herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgG₁ human constantdomain, see, e.g., international applications WO02/060955 andWO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgG₁ constantregion.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art,see, e.g., U.S. Pat. No. 5,837,821 or international application WO94/09817.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase αSyn localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement binding) to be modulated.Such partial deletions of the constant regions may improve selectedcharacteristics of the antibody (serum half-life) while leaving otherdesirable functions associated with the subject constant region domainintact. Moreover, as alluded to above, the constant regions of thedisclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it may be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to aggregate αSyn. Standard techniques known tothose of skill in the art can be used to introduce mutations in thenucleotide sequence encoding an antibody, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference V_(H) region,V_(H)-CDR1, V_(H)-CDR2, V_(H)-CDR3, V_(L) region, V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind aggregate αSyn).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen-binding activity or alteration in binding activity (e.g.,improvements in antigen-binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of aggregate αSyn) can be determined using techniques describedherein or by routinely modifying techniques known in the art.

Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an antibody, orantigen-binding fragment, variant, or derivative thereof can be composedof triple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original B cells,hybridoma cells or from other transformed cells by standard techniques,such as guanidinium isothiocyanate extraction and precipitation followedby centrifugation or chromatography. Where desirable, mRNA may beisolated from total RNA by standard techniques such as chromatography onoligo dT cellulose. Suitable techniques are familiar in the art. In oneembodiment, cDNAs that encode the light and the heavy chains of theantibody may be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well-known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as human constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)), where at least one of the CDRs of the heavy chain variableregion or at least two of the V_(H)-CDRs of the heavy chain variableregion are at least 80%, 85%, 90% or 95% identical to reference heavychain V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Alternatively, the V_(H)-CDR1,V_(H)-CDR2, or V_(H)-CDR3 regions of the V_(H) are at least 80%, 85%,90% or 95% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a heavy chain variable regionof the invention has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences of the antibodiesillustrated in the Examples.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(V_(L)), where at least one of the V_(L)-CDRs of the light chainvariable region or at least two of the V_(L)-CDRs of the light chainvariable region are at least 80%, 85%, 90% or 95% identical to referencelight chain V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 amino acid sequencesfrom the antibodies disclosed herein. Alternatively, the V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3 regions of the V_(L) are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2,and V_(L)-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a light chain variable regionof the invention has V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 polypeptidesequences related to the polypeptide sequences of the antibodiesillustrated in the Examples.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally, polynucleotideswhich encode fusion polynucleotides, Fab fragments, scFvs fragments, andother derivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides, e.g., as described in Kutmeieret al., BioTechniques 17 (1994), 242, which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA⁺ RNA, isolated from, any tissue or cellsexpressing the aggregate αSyn-specific antibody, such as hybridoma cellsselected to express an antibody) by PCR amplification using syntheticprimers hybridizable to the 3′ and 5′ ends of the sequence or by cloningusing an oligonucleotide probe specific for the particular gene sequenceto identify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule is described herein. Once a polynucleotide encoding anantibody molecule or a heavy or light chain of an antibody, or portionthereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,international applications WO 86/05807 and WO 89/01036; and U.S. Pat.No. 5,122,464) and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells. For the purposes of this invention, numerousexpression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Othersinvolve the use of polycistronic systems with internal ribosome bindingsites. Additionally, cells which have integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow selection of transfected host cells. The marker may provide forprototrophy to an auxotrophic host, biocide resistance (e.g.,antibiotics) or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals.

In some embodiments the cloned variable region genes are inserted intoan expression vector along with the heavy and light chain constantregion genes as discussed above. In one embodiment, this is affectedusing a proprietary expression vector of Biogen IDEC, Inc., referred toas NEOSPLA, disclosed in U.S. Pat. No. 6,159,730. This vector containsthe cytomegalovirus promoter/enhancer, the mouse beta globin majorpromoter, the SV40 origin of replication, the bovine growth hormonepolyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2,the dihydrofolate reductase gene and leader sequence. This vector hasbeen found to result in very high-level expression of antibodies uponincorporation of variable and constant region genes, transfection in CHOcells, followed by selection in G418 containing medium and methotrexateamplification. Of course, any expression vector which is capable ofeliciting expression in eukaryotic cells may be used in the presentinvention. Examples of suitable vectors include, but are not limited toplasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (availablefrom Invitrogen, San Diego, Calif.), and plasmid pCI (available fromPromega, Madison, Wis.). In general, screening large numbers oftransformed cells for those which express suitably high levels ifimmunoglobulin heavy and light chains is routine experimentation whichcan be carried out, for example, by robotic systems. Vector systems arealso taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which isincorporated by reference in its entirety herein. This system providesfor high expression levels, e.g., >30 pg/cell/day. Other exemplaryvector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.

In other embodiments the antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be expressed usingpolycistronic constructs such as those disclosed in US patentapplication publication no. 2003-0157641 A1 and incorporated herein inits entirety. In these expression systems, multiple gene products ofinterest such as heavy and light chains of antibodies may be producedfrom a single polycistronic construct. These systems advantageously usean internal ribosome entry site (IRES) to provide relatively high levelsof antibodies. Compatible IRES sequences are disclosed in U.S. Pat. No.6,193,980 which is also incorporated herein. Those skilled in the artwill appreciate that such expression systems may be used to effectivelyproduce the full range of antibodies disclosed in the instantapplication.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection including lipotransfection using, e.g., Fugene orlipofectamine, protoplast fusion, calcium phosphate precipitation, cellfusion with enveloped DNA, microinjection, and infection with intactvirus. Typically, plasmid introduction into the host is via standardcalcium phosphate co-precipitation method. The host cells harboring theexpression construct are grown under conditions appropriate to theproduction of the light chains and heavy chains and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),enzyme immuno assay (EIA) or fluorescence-activated cell sorter analysis(FACS), immunohistochemistry and the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, NSO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese Hamster Ovary (CHO) cells, in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies;see, e.g., Foecking et al., Gene 45 (1986), 101; Cockett et al.,Bio/Technology 8 (1990), 2.

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3×63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO and 293 cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, N Y (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, N Y(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification, for a review, see Bebbington and Hentschel. The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3(1983), 257.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-) affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can also be expressed innon-mammalian cells such as bacteria or insect or yeast or plant cells.Bacteria which readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies; see, e.g., internationalapplication WO02/096948.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983),1791), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13(1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989),5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrix ofglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP1 gene which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example ATCC No. 44076 orPEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trp1 lesionas a characteristic of the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingfor example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,e.g. ammonium sulfate precipitation, or by any other standard techniquefor the purification of proteins; see, e.g., Scopes, “ProteinPurification”, Springer Verlag, N.Y. (1982). Alternatively, a preferredmethod for increasing the affinity of antibodies of the invention isdisclosed in US patent publication 2002-0123057 A1.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain Fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinaggregate αSyn-binding domain with at least one target binding site, andat least one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species.

As discussed in more detail elsewhere herein, antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, antibodies may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins; see, e.g., international applicationsWO92/08495; WO91/14438; WO89/12624; U.S. Pat. No. 5,314,995; andEuropean patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from post-translation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, e.g., Proteins—Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of or consists of, apolypeptide having the amino acid sequence of any one or more of theV_(H) regions of an antibody of the invention or the amino acid sequenceof any one or more of the V_(L) regions of an antibody of the inventionor fragments or variants thereof, and a heterologous polypeptidesequence. In another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises, consistsessentially of, or consists of a polypeptide having the amino acidsequence of any one, two, three of the V_(H)-CDRs of an antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the V_(L)-CDRs of an antibody, or fragments,variants, or derivatives thereof, and a heterologous polypeptidesequence. In one embodiment, the fusion protein comprises a polypeptidehaving the amino acid sequence of a V_(H)-CDR3 of an antibody of thepresent invention, or fragment, derivative, or variant thereof, and aheterologous polypeptide sequence, which fusion protein specificallybinds to αSyn. In another embodiment, a fusion protein comprises apolypeptide having the amino acid sequence of at least one V_(H) regionof an antibody of the invention and the amino acid sequence of at leastone V_(L) region of an antibody of the invention or fragments,derivatives or variants thereof, and a heterologous polypeptidesequence. Preferably, the V_(H) and V_(L) regions of the fusion proteincorrespond to a single source antibody (or scFv or Fab fragment) whichspecifically binds aggregate αSyn. In yet another embodiment, a fusionprotein for use in the diagnostic and treatment methods disclosed hereincomprises a polypeptide having the amino acid sequence of any one, two,three or more of the V_(H) CDRs of an antibody and the amino acidsequence of any one, two, three or more of the V_(L) CDRs of anantibody, or fragments or variants thereof, and a heterologouspolypeptide sequence. Preferably, two, three, four, five, six, or moreof the V_(H)-CDR(s) or V_(L)-CDR(s) correspond to single source antibody(or scFv or Fab fragment) of the invention. Nucleic acid moleculesencoding these fusion proteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA CellBiol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991), 721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half-life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo; see, e.g., Leonget al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002),531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide (HIS), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767)and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made may be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular, antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared e.g.by reacting an αSyn binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g. those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, demonstrate presence of a neurologicaldisease, to indicate the risk of getting a neurological disease, tomonitor the development or progression of a neurological disease, i.e.synucleinopathic disease as part of a clinical testing procedure to,e.g., determine the efficacy of a given treatment and/or preventionregimen. Detection can be facilitated by coupling the antibody, orantigen-binding fragment, variant, or derivative thereof to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions; see, e.g., U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude. ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”Microbiological Associates Quarterly Publication, Walkersville, Md.,Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol. 31(1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E.(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo(1981). The enzyme, which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985), and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

Compositions and Methods of Use

The present invention relates to compositions comprising theaforementioned αSyn aggregate binding molecule, e.g., antibody orantigen-binding fragment thereof of the present invention or derivativeor variant thereof, or the polynucleotide, vector or cell of theinvention for the use in an early diagnostic or therapeutic. Acomposition of the present invention may further comprise apharmaceutically acceptable carrier. Furthermore, the pharmaceuticalcomposition of the present invention may comprise further agents such asinterleukins or interferons depending on the intended use of thepharmaceutical composition. For example, for use in the treatment ofParkinson's disease the additional agent may be selected from the groupconsisting of small organic molecules, anti-αSyn aggregate antibodies,and combinations thereof. Hence, in a particular preferred embodimentthe present invention relates to the use of the αSyn binding molecule,e.g., antibody or antigen-binding fragment thereof of the presentinvention or of a binding molecule having substantially the same bindingspecificities of any one thereof, the polynucleotide, the vector or thecell of the present invention for the preparation of a pharmaceutical ordiagnostic composition for prophylactic and therapeutic treatment of asynucleinopathic disease, monitoring the progression of asynucleinopathic disease or a response to a synucleinopathic diseasetreatment in a subject or for determining a subject's risk fordeveloping a synucleinopathic disease.

Hence, in one embodiment the present invention relates to a method oftreating a neurological disorder characterized by abnormal accumulationand/or deposition of αSyn in the brain and the central nervous system,respectively, which method comprises administering to a subject in needthereof a therapeutically effective amount of any one of theafore-described aggregate αSyn binding molecules, antibodies,polynucleotides, vectors or cells of the instant invention. The term“neurological disorder” includes but is not limited to synucleinopathicdiseases such as Parkinson's disease (PD), Parkinson's disease dementia(PDD), dementia with Lewy bodies (DLB), the Lewy body variant ofAlzheimer's disease (LBVAD), multiple systems atrophy (MSA), pureautonomic failure (PAF), neurodegeneration with brain iron accumulationtype-1 (NBIA-I), Alzheimer's disease, Pick disease, juvenile-onsetgeneralized neuroaxonal dystrophy (Hallervorden-Spatz disease),amyotrophic lateral sclerosis, traumatic brain injury, and Down syndromeas well as other movement disorders and disease of the central nervoussystem (CNS) in general. Unless stated otherwise, the termsneurodegenerative, neurological or neuropsychiatric are usedinterchangeably herein.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that the antibodies of the present inventionhave a high specificity, or are even specific to, aggregates of αSynwith little or no specificity to the physiological monomer of αSyn.Therefore, the antibodies of the present invention are capable ofpreventing a clinically manifest synucleinopathic disease, or ofdiminishing the risk of the occurrence of the clinically manifestdisease, or of delaying the onset or progression of the clinicallymanifest disease.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-αSyn antibody,binding fragment, derivative or variant thereof, polynucleotide, vectoror cell of the present invention. Associated with such container(s) canbe a notice in the form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, or alternatively, the kitcomprises reagents and/or instructions for use in appropriate diagnosticassays. The composition, e.g. kit of the present invention is of courseparticularly suitable for the risk assessment, diagnosis, prevention andtreatment of a disorder which is accompanied with the presence of αSyn,and in particular applicable for the treatment of Parkinson's disease(PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies(DLB) and Lewy body variant of Alzheimer's disease (LBVAD).

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be affected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, intranasal,topical or intradermal administration or spinal or brain delivery.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are preferably adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal administration may be presented as asuppository with a suitable carrier.

Furthermore, whereas the present invention includes the now standardprocedure of drilling a small hole in the skull to administer a drug ofthe present invention, in a preferred aspect, the binding molecule,especially antibody or antibody-based drug of the present invention cancross the blood-brain barrier, which allows for intravenous or oraladministration.

In further embodiment, the composition further includes loading the oneor more of the above described ingredients, e.g. anti-αSyn antibody,binding fragment, derivative or variant thereof, polynucleotide, vectoror cell of the present invention, into a nanoparticle carrier. Thenanoparticle may be any known in the art, for example polyanhydridenanoparticles. The nanoparticles may help to increase the half-life ofthe compositions from preventing them leaking out of the vasculature orbeing taken up into off site targets. The nanoparticles may also aid inthe transition through the blood brain barrier and help target theingredients to their intended sites. Using nanoparticles may allow alower dosage of the ingredients due to these benefits provided by anincreased half-life and better targeting. The nanoparticles may furtherbe functionalized by conjugating with various materials, such as PEG.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example, dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimensentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

Furthermore, in a preferred embodiment of the present invention thepharmaceutical composition may be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises ananti-αSyn aggregate antibody or binding fragment, derivative or variantthereof for passive immunization. As mentioned in the backgroundsection, oligomeric species of α-synuclein have been reportedextracellularly in plasma and CSF (El-Agnaf et al., FASEB J. 20 (2006),419-425) and passive immunization studies in mouse models of Parkinson'sdisease show that extracellular mouse monoclonal antibodies againstα-synuclein can reduce accumulation of intracellular α-synucleinaggregates (Masliah et al., Neuron, 46 (2005), 857-868). Accordingly, itis prudent to expect that the human anti-αSyn aggregate antibodies andequivalent αSyn binding molecules of the present invention areparticularly useful as a vaccine for the prevention or amelioration ofsynucleinopathic diseases such as PD, DLB and LBVAD.

In one embodiment, it may be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the presentinvention, which might more readily penetrate a cell membrane. Forexample, Robert et al., Protein Eng. Des. Sel. (2008) October 16;S1741-0134, published online ahead, describe the use of chimericrecombinant Fab (rFab) and single chain fragments (scFvs) of monoclonalantibody WO-2 which recognizes an epitope in the N-terminal region ofAP. The engineered fragments were able to (i) prevent amyloidfibrillization, (ii) disaggregate preformed Aβ1-42 fibrils and (iii)inhibit Aβ1-42 oligomer-mediated neurotoxicity in vitro as efficientlyas the whole IgG molecule. The perceived advantages of using small Faband scFv engineered antibody formats which lack the effector functioninclude more efficient passage across the blood-brain barrier andminimizing the risk of triggering inflammatory side reactions.Furthermore, besides scFv and single-domain antibodies retain thebinding specificity of full-length antibodies, they can be expressed assingle genes and intracellularly in mammalian cells as intrabodies, withthe potential for alteration of the folding, interactions,modifications, or subcellular localization of their targets; see forreview, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called “SuperAntibodyTechnology”, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term “TransMabs” has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other neuroprotective agents useful for treating a synucleinopathicdisease may be desirable. In one embodiment, the additional agent iscomprised in the pharmaceutical composition of the present invention.Examples of neuroprotective agents which can be used to treat a subjectinclude, but are not limited to, an acetylcholinesterase inhibitor, aglutamatergic receptor antagonist, kinase inhibitors, HDAC inhibitors,anti-inflammatory agents, divalproex sodium, or any combination thereof.Examples of other neuroprotective agents that may be used concomitantwith pharmaceutical composition of the present invention are describedin the art; see, e.g. international application WO2007/011907. In oneembodiment, the additional agent is dopamine or a dopamine receptoragonist.

In a further embodiment of the present invention the αSyn bindingmolecules, in particular antibodies of the present invention, may alsobe co-administered or administered before or after transplantationtherapy with neural transplants or stem cell therapy useful for treatinga synucleinopathic disease. Such approaches with transplants ofembryonic mesencephalic neurons have been performed in patients withParkinson's disease with the aim of replacing the neurons that are lostin the disease and reinstating dopaminergic neurotransmission in thestriatum. After 11-16 years post transplantation, the grafted neuronswere found to contain Lewy bodies and Lewy neurites. This spread of αSynpathology from the host to the grated tissues may be prevented byco-administration of αSyn binding molecules, in particular antibodies ofthe present invention.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Preferably, thetherapeutic agent in the composition is present in an amount sufficientto restore or preserve normal behavior and/or cognitive properties incase of PD, DLB or other synucleinopathic diseases.

From the foregoing, it is evident that the present invention encompassesany use of an αSyn binding molecule comprising at least one CDR of theabove described antibody, in particular for diagnosing and/or treatmentof a synucleinopathic disease as mentioned above, particularlyParkinson's disease. Preferably, said binding molecule is an antibody ofthe present invention or an immunoglobulin chain thereof. In addition,the present invention relates to anti-idiotypic antibodies of any one ofthe mentioned antibodies described hereinbefore. These are antibodies orother binding molecules which bind to the unique antigenic peptidesequence located on an antibody's variable region near theantigen-binding site and are useful, e.g., for the detection ofanti-αSyn antibodies in sample of a subject.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described αSyn bindingmolecules, antibodies, antigen-binding fragments, polynucleotides,vectors or cells of the invention and optionally suitable means fordetection such as reagents conventionally used in immuno or nucleic acidbased diagnostic methods. The antibodies of the invention are, forexample, suited for use in immunoassays in which they can be utilized inliquid phase or bound to a solid phase carrier. Examples of immunoassayswhich can utilize the antibody of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA), thesandwich (immunometric assay), such as enzyme immuno assay (EIA), flowcytometry and the Western blot assay. The antigens and antibodies of theinvention can be bound to many different carriers and used to isolatecells specifically bound thereto. Examples of well-known carriersinclude glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amyloses, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite.Nanoparticles may also be used as a carrier. The nature of the carriercan be either soluble or insoluble for the purposes of the invention.There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,colloidal metals, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds; see also the embodiments discussedhereinabove.

By a further embodiment, the αSyn binding molecules, in particularantibodies of the present invention may also be used in a method for thediagnosis of a disorder in an individual by obtaining a body fluidsample from the tested individual which may be a blood sample, a lymphsample or any other body fluid sample and contacting the body fluidsample with an antibody of the instant invention under conditionsenabling the formation of antibody-antigen complexes. The level of suchcomplexes is then determined by methods known in the art, a levelsignificantly higher than that formed in a control sample indicating thedisease in the tested individual. In the same manner, the specificantigen bound by the antibodies of the invention may also be used. Thus,the present invention relates to an in vitro immunoassay comprising thebinding molecule, e.g., antibody or antigen-binding fragment thereof ofthe invention as illustrated in the Examples.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, an antibody-basedarray may be used, which is for example loaded with antibodies orequivalent antigen-binding molecules of the present invention whichspecifically recognize αSyn. Design of microarray immunoassays issummarized in Kusnezow et al., Mol. Cell Protcomics 5 (2006), 1681-1696.Accordingly, the present invention also relates to microarrays loadedwith αSyn binding molecules identified in accordance with the presentinvention.

In one embodiment, the present invention relates to a method ofdiagnosing a synucleinopathic disease in a subject, the methodcomprising: (a) assessing a level of αSyn in a sample from the subjectto be diagnosed with an antibody of the present invention, an αSynbinding fragment thereof or an αSyn binding molecule havingsubstantially the same binding specificities of any one thereof; and (b)comparing the level of the αSyn to a reference standard that indicatesthe level of the αSyn in one or more control subjects, wherein adifference or similarity between the level of the αSyn and the referencestandard indicates that the subject has Parkinson's disease.

The subject to be diagnosed may be asymptomatic or preclinical for thedisease. Preferably, the control subject has a synucleinopathic disease,for example PD, DLB or LBVAD, wherein a similarity between the level ofαSyn and the reference standard indicates that the subject to bediagnosed has a synucleinopathic disease. Alternatively, or in additionas a second control the control subject does not have a synucleinopathicdisease, wherein a difference between the level of αSyn and thereference standard indicates that the subject to be diagnosed has asynucleinopathic disease. Preferably, the subject to be diagnosed andthe control subject(s) are age-matched. The sample to be analyzed may beany body fluid suspected to contain αSyn, for example a blood, or afraction of blood, CSF, or urine sample

The level of α-synuclein may be assessed by any suitable method known inthe art comprising, e.g., analyzing αSyn by one or more techniqueschosen from enzyme immuno asay (EIA), Western blot, immunoprecipitation,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),fluorescent activated cell sorting (FACS), two-dimensional gelelectrophoresis, mass spectroscopy (MS), matrix-assisted laserdesorption/ionization-time of flight-MS (MALDI-TOF), surface-enhancedlaser desorption ionization-time of flight (SELDI-TOF), high performanceliquid chromatography (HPLC), fast protein liquid chromatography (FPLC),multidimensional liquid chromatography (LC) followed by tandem massspectrometry (MS/MS), and laser densitometry. Preferably, said in vivoimaging of αSyn comprises positron emission tomography (PET), singlephoton emission tomography (SPECT), near infrared (NIR) optical imagingor magnetic resonance imaging (MRI).

Methods of diagnosing a synucleinopathic disease such as Parkinson'sdisease or Lewy body disease, monitoring a synucleinopathic diseaseprogression, and monitoring a synucleinopathic disease treatment usingantibodies and related means which may be adapted in accordance with thepresent invention are also described in international applicationWO2007/011907. Similarly, antibody-based detection methods for αSyn aredescribed in international applications WO99/50300, WO2005/047860,WO2007/021255 and WO2008/103472, the disclosure content of all beingincorporated herein by reference. Those methods may be applied asdescribed but with an αSyn aggregate specific antibody, bindingfragment, derivative or variant of the present invention.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example, the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES Example 1

In order to generate antibodies specific to the toxic aggregates ofhuman αSyn, both polyclonal and monoclonal antibodies were generated.The polyclonal antibodies were generated by first aggregating nativehuman αSyn in vitro and then using the aggregates to immunize mice. Themice were injected with a primary injection followed by three boosterdoses admixed with an alum adjuvant. Mouse sera was then selectivelyenriched for the HA-PAbs through the depletion of antibodies which boundto targets other than the aggregated human αSyn. The monoclonalantibodies were generated using murine hybridomas created by the fusionof murine splenic plasma cells with myeloma cells.

The HA-PAbs were screen by exposing them to epitopes unique to nativeαSyn, epitopes common to αSyn and βSyn, and epitopes shared betweennative αSyn and toxic αSyn. As shown by the different intensities inFIG. 1, the toxic aggregated αSyn has unique epitopes that are absent inthe physiological monomeric form of αSyn. This allows the screen forHA-PAbs that may distinguish between the two forms and to select forthose antibodies which have a high affinity for only the toxic aggregateof αSyn. As shown in FIG. 2, when comparing the screen antibodies tocommercially available antibodies, the HA-PAbs have a high affinity foronly the aggregate form, but not to either BSA or the pure monomer formof αSyn, whereas all the commercially available antibodies showed nopreference for either form, though they still did not bind to BSA.

Twenty-six hybridoma clones were created which had HM-MAbs with a highspecificity to only the aggregated form of αSyn. As shown in FIG. 4,when comparing the blot of the monomeric form with an aggregatedrecombinant αSyn, the HM-MAbs, none of the monomeric form could bedetected by immunoblot indicated a lack of binding affinity for thisform of αSyn by the HM-MAbs. Of these 26 hybridomas, 21 clones wereconfirmed to secrete a single antibody isotype.

Therefore, it is possible to identify and select for antibodies whichhave a high affinity for only the aggregate form of αSyn. Theseantibodies may then be used to detect the aggregate form in subjects toaid in the diagnostic of αSyn aggregate diseases, such as Parkinson'sdisease.

Example 2

To show that the HM-PAbs of Example 1 can discriminate between subjectswith Parkinson's disease (PD) and subjects which were age matched (AM)an immunoblot comparison was carried out. Serum and cerebral spinalfluid (CSF) samples were taken from both PD patients and AM subjects.The protein was then extracted from all the samples and using animmunoblot with HM-PAbs as the primary antibody, it was shown that theantibodies could detect the aggregate form in both the CSF and sera ofthe subjects (see FIG. 3).

Additionally, the HM-PAbs showed that PD patients contained a higheramount of aggregate αSyn in their CSF for every comparison with AMsubjects (see FIG. 3). This discriminatory ability of the HM-PAbs may beused as an early detection test for aiding in the diagnostic of subjectsfor αSyn aggregate diseases, such as PD, LB dementia, and othersynucleoinopathies.

Therefore, it is possible to specifically detect aggregate αSyn in boththe CSF and sera of subjects, with the CSF providing a good sample touse for diagnosis subjects with PD, and other synucleoinopathies, usinghighly specific HM-PAbs.

Example 3

The aggregate specific HM-MAbs of Example 1 also show an ability todiscriminate between patients with PD and AM subjects. Sera samples weretaken from patients with PD and AM subjects and the protein isolated inorder to determine if the HM-MAbs could detect aggregate αSyn in asubject's sample. As shown in FIG. 4, the HM-MAbs were able to detectaggregate αSyn in the sera of both patients with PD and AM subjects.

To show that the HM-MAbs have diagnostics ability, an indirect,competitive semi-quantitative Enzyme Immuno Assay (EIA) was developed.To first show the ability of the EIA to distinguish between aggregateand monomeric forms of αSyn, aggregate EIA was immobilized to the EIAplate and then either the monomer or aggregate αSyn was added as a freeanalyte to compete for the primary antibody. The IgM HM-MAbs fromhybridoma strain 3A8 was then added to each well and allowed to bindwith the immobilized aggregate αSyn or to the free analyte. The EIAplate was then washed to remove the free analyte and any bound antibody.The fixed aggregate αSyn and any bound antibody was then further boundto a secondary antibody conjugated with an enzyme to produce light whendeveloped. As shown in FIG. 5A, free aggregated αSyn showed a dosedependent decrease in the optical density in the EIA. This shows thatthe free aggregate αSyn competed for primary antibody. However, themonomer/linear form of αSyn did not show any decrease in optical densityof the EIA, lacking the ability to compete for the HM-MAb. This showsthat the EIA using the HM-MAb from 3A8 is selective for only theaggregate form of αSyn.

To show that the 3A8 HM-MAb could also discriminate between PD patientsand AM subjects, the sera samples from the patients and subjects werelikewise compared. Aggregate αSyn was immobilized as above, but the seraat either a 1:200 or a 1:400 dilution from either the PD patients or theAM subjects was used as the free analyte. As shown in FIG. 5B, foreither dilution factor, the PD patients showed about twice thecompetition for the HM-MAb than the AM control subjects. Therefore, the3A8 HM-MAb shows that using the indirect, competitive semi-quantitativeEIA test is able to discriminate between individuals with PD andcontrols.

Due to its selective affinity for the aggregate form of αSyn, the HM-MAbcan be used to aid in the early diagnosis of individuals with PD, andother synucleoinopathies.

Example 4

To further characterize and create novel diagnostics andimmunotherapeutics for PD the HM-MAbs, two hybridoma clones wereselected for sequencing and recombinant scFvs generation, 3A8 and 6G7.The hybridoma clone 3A8 secreted monoclonal antibodies of the IgMisotype and kappa light chains and clone 6G7 secreted monoclonalantibodies of the IgG isotype and kappa light chains).

To generate recombinant scFvs from 3A8 and 6G7 for development of noveldiagnostics and nano-immunotherapeutics for Parkinson's disease, totalcellular RNA was first isolated. Total RNA from hybridoma cells of 3A8and 6G7 were prepared using RNeasy Mini Kit (Qiagen) according to theinstructions of the manufacturer. Specifically, 5×10⁵ viable hybridomacells were washed with DEPC-treated water, lysed and total RNA isolatedas per the instructions of the manufacturer. Total RNA was quantitatedand then confirmed by denaturing agarose gel electrophoresis andvisualization of the 28S and 18S rRNA under UV (FIG. 6).

Next, cDNA was synthesized from total cellular RNA from each of the twohybridoma clones using the SuperScript® III One-Step RT-PCR System(Invitrogen™) as instructed by the manufacturer. The cDNA from each ofthe two hybridomas then served as the template for PCR amplification ofthe cognate variable heavy chain and light chains using a set ofoligonucleotide primers designed as described previously (Yuan et al.2004, A simple and rapid protocol for the sequence determination offunctional kappa light chain cDNAs from aberrant-chain-positive murinehybridomas. Journal of Immunological Methods 294(1-2):199-207doi:10.1016/j.jim.2004.09.001, herein incorporated by reference).Amplicons were visualized under UV (FIG. 7), purified using the QIAquickPCR Purification Kit (Qiagen) as per the kit instructions, quantitatedand then ligated into the pCRTM2.1 TA cloning vector (ThermoFisherScientific). The ligated plasmid DNA was transformed via heat shock intochemically competent Escherichia coli host strains and plated on mediacontaining the appropriate antibiotic.

Following overnight growth of the transformed E. coli at 37° C., tworecombinant colonies, harboring plasmids carrying putativeantibody-encoding genes from hybridoma clones 3A8 and 6G7, were expandedand purified plasmids from such bacterial clones were subjected to DNAsequencing in both directions. The amino acid sequences were deducedfrom the respective nucleotide sequences using Snap software. Theantibody sequences were designated complementarity determining regions(CDRs) of light and heavy chains of the isolated immunoglobulin genesusing Kabat database (FIGs. for 3A8 and FIGs. for 6G7 summarized inTable 2).

TABLE 2 Amino acid sequences for the variable regions ofthe heavy-chain (VH) and light-chain (LV) of the3A8 and 6G7. Denotation of complementarity-determining region (CDR) sequence alignment of 3A8and 6G7 antibody. Amino acid sequences werededuced from DNA sequences CDRs were selected asdescribed in Kabat data base. Clone 3A8 Clone 6G7 Light Chain CDR1SGNIHNYLA SGNIHNYLA (SEQ ID NO: 13) (SEQ ID NO: 31) CDR2 NAKTLADNAKTLAD  (SEQ ID NO: 15) (SEQ ID NO: 33) CDR3 QHFWSTPWT QHFWSTPWT(SEQ ID NO: 17) (SEQ ID NO: 35) Heavy Chain CDR1 GFTFSSY GFTFSNY(SEQ ID NO: 4) (SEQ ID NO: 22) CDR2 SSGGSY RLKSNNYATHYA (SEQ ID NO: 6)(SEQ ID NO: 24) CDR3 DGVWLRPYYFDY LLRP (SEQ ID NO: 8) (SEQ ID NO: 26)

Surprisingly, upon DNA sequencing, it was determined that the 3A8 and6G7 monoclonal antibodies are encoded by disparate (geneticallydifferent) variable heavy chains but share the same light chain. Thepresence of two separate heavy chains and, therefore, two separateantigen receptors on a single B cell may have ramifications for bothpolyclonal activation and toleration of human recombinantα-Synuclein-specific B cells.

The classification of germline-based 3A8 and 6G7 antibodies variableregion sequence was identified from the IMGT (the internationalImMunoGeneTics information system (http://www.imgt.org) database. Theclassification of germline-based 3A8 antibody variable region sequencewas identified from the IMGT database. The variable light chain of the3A8 antibody gene belonged to the immunoglobulin mouse kappa,IGV_(κ)V_(XII) (IG_(κ)V₁₂) and contained IGKJ₁ gene segments. Thevariable heavy chain belonged to the immunoglobulin mouse VH_(V) (IGVH₅)subgroup gene family with JH₂ and D₂ segments. The classification ofgermline-based 6G7 antibody variable region sequence was identified fromthe IMGT database. The variable light chain of the 6G7 antibody genebelonged to the immunoglobulin mouse kappa, IGV_(κ)V_(XII) (IG_(κ)V₁₂)and contained IGKJ₁ gene segments. The variable heavy chain belonged tothe immunoglobulin mouse VH VI (IGVH6) subgroup gene family with JH₂ andD₁ segments.

Example 5 Clone 3A8

3A8 Heavy chain (SEQ ID NO: 2)        10        20        30        40        50 Numbering1234567890123456789012345678901234567890123456789012a3456789 Mouse 3A8EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGSYTYY60        70        80           90                110   11301234567890123456789012abc345678901234567891abcd234567890123 Mouse 3A8PDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRDGVWLRPYYFDYWGQGTTVTVS

TABLE 3 Sequence of mouse 3A8 variable heavy chainVariable 3A8 heavy chain sequence denotation HFR1EVMLVESGGGLVKPGGSLKLSCAAS (SEQ ID NO: 3) HCDR1 GFTFSSY (SEQ ID NO: 4)HFR2 TMSWVRQTPEKRLEWVATI (SEQ ID NO: 5) HCDR2 SSGGSY (SEQ ID NO: 6) HFR3TYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTR (SEQ ID NO: 7) HCDR3DGVWLRPYYFDY (SEQ ID NO: 8) HFR4 WGQGTTVTVS (SEQ ID NO: 9)

>3A8 variable heavy chain DNA nucleotide sequence (SEQ ID NO: 10)GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATGGGGTATGGTTACGCCCGTACTACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCC >3A8 variable heavy chain Protein Sequence(SEQ ID NO: 2)EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRDGVWLRPYYFDYWGQGTTVTVS >3A8 variable heavy chain DNA nucleotide (SEQ ID NO: 10) and encodedprotein sequence (SEQ ID NO: 2) 1 E  V  M  L  V  E  S  G  G  G  L  V  K  P  G  G  S  L  K  L 20 1GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTC 60 21 S  C  A  A  S  G  F  T  F  S  S  Y  T  M  S  W  V  R  Q  T 40 61TCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATACCATGTCTTGGGTTCGCCAGACT 120 41 P  E  K  R  L  E  W  V  A  T  I  S  S  G  G  S  Y  T  Y  Y 60 121CCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTACTAT 180 61 P  D  S  V  K  G  R  F  T  I  S  R  D  N  A  K  N  T  L  Y 80 181CCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTAC 240 81 L  Q  M  S  S  L  K  S  E  D  T  A  M  Y  Y  C  T  R  D  G 100 241CTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATGGG 300 101 V  W  L  R  P  Y  Y  F  D  Y  W  G  Q  G  T  T  V  T  V  S 120 301GTATGGTTACGCCCGTACTACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCC 3603A8 variable Light chain (SEQ ID NO: 11)        10        20        30        40         50 Numbering12345678901234567890123456789012345678901234567890123456789 Mouse 3A8DILMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVP60        70        80        90        100   1080123456789012345678901234567890123456789012345678 Mouse 3A8SRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEMKR

TABLE 4 Sequence of mouse 3A8 variable heavy chainVariable 3A8 light chain sequence denotation LFR1DILMTQSPASLSASVGETVTITCRA (SEQ ID NO: 12) LCDR1 SGNIHNYLA(SEQ ID NO: 13) LFR2 WYQQKQGKSPQLLVY (SEQ ID NO: 14) LCDR2 NAKTLAD(SEQ ID NO: 15) LFR3 GVPSRFSGSGSGTQYSLKINSLQPEDFGSYYC (SEQ ID NO: 16)LCDR3 QHFWSTPWT (SEQ ID NO: 17) LFR4 FGGGTKLEMKR (SEQ ID NO: 18)

Light Chain Sequence

>3A8 light chain DNA nucleotide Sequence (SEQ ID NO: 19)GACATTCTGATGACCCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATGAAACGC >3A8 light chain protein Sequence (SEQ ID NO: 11)DILMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEMKR >3A8 light chain DNA nucleotide Sequence (SEQ ID NO: 19) and encodedprotein sequence (SEQ ID NO: 11) 1 D  I  L  M  T  Q  S  P  A  S  L  S  A  S  V  G  E  T  V  T 20 1GACATTCTGATGACCCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACC 60 21 I  T  C  R  A  S  G  N  I  H  N  Y  L  A  W  Y  Q  Q  K  Q 40 61ATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAG 120 41 G  K  S  P  Q  L  L  V  Y  N  A  K  T  L  A  D  G  V  P  S 60 121GGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCA 180 61 R  F  S  G  S  G  S  G  T  Q  Y  S  L  K  I  N  S  L  Q  P 80 181AGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCT 240 81 E  D  F  G  S  Y  Y  C  Q  H  F  W  S  T  P  W  T  F  G  G 100 241GAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGTGGACGTTCGGTGGA 300 101 G  T  K  L  E  M  K  R 108 301 GGCACCAAGCTGGAAATGAAACGC 324

Clone 6G7

>6G7 variable Heavy chain (SEQ ID NO: 20)         10        20        30        40        50 Numbering1234567890123456789012345678901234567890123456789012abc34567 6G7DVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYAT   60        70        80           90     111         1128901234567890123456789012abc34567890123456123456789101112 Mouse 6G7HYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRLLRPWGQGTTLTVS

Variable 6G7 heavy chain sequence denotation HFR1DVQLQQSGGGLVQPGGSMKLSCVAS (SEQ ID NO: 21) HCDR1 GFTFSNY (SEQ ID NO: 22)HFR2 WMNWVRQSPEKGLEWVAEI (SEQ ID NO: 23) HCDR2RLKSNNYATHYA (SEQ ID NO: 24) HFR3 ESVKGRFTISRDDSKSSVYLQMNNLRAEDIGIYYCIR(SEQ ID NO: 25) HCDR3 LLRP (SEQ ID NO: 26) HFR4WGQGTTLTVS (SEQ ID NO: 27)

Heavy Chain of 6G7 Clone

>6G7 Heavy chain DNA nucleotide sequence (SEQ ID NO: 28)GATGTGCAGCTTCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGTTACTACGGCCCTGGGGCCAAGGCACCACTCTCACAGTCTCCTC >6G7 Heavy chain protein sequence(SEQ ID NO: 20)DVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRLLRPWGQGTTLTVS >6G7 heavy chain protein (SEQ ID NO: 20) and DNA nucleotide Sequence (SEQID NO: 28)   1 D  V  Q  L  Q  Q  S  G  G  G  L  V  Q  P  G  G  S  M  K  L  20  82GATGTGCAGCTTCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTC 141  21 S  C  V  A  S  G  F  T  F  S  N  Y  W  M  N  W  V  R  Q  S  40 142TCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCT 201  41 P  E  K  G  L  E  W  V  A  E  I  R  L  K  S  N  N  Y  A  T  60 202CCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACA 261  61 H  Y  A  E  S  V  K  G  R  F  T  I  S  R  D  D  S  K  S  S  80 262CATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGT 321  81 V  Y  L  Q  M  N  N  L  R  A  E  D  T  G  I  Y  Y  C  T  R 100 322GTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGG 381 101 L  L  R  P  W  G  Q  G  T  T  L  T  V  S 114 382TTACTACGGCCCTGGGGCCAAGGCACCACTCTCACAGTCTCC 423

6G7 Light Chain (SEQ ID NO: 29)

        10        20        30        40        50 Numbering12345678901234567890123456789012345678901234567890123456789 Mouse 6G7DILMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVP60        70        80        90       100    1080123456789012345678901234567890123456789012345678 Mouse 6G7SRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEMKR

Variable 6G7 light chain sequence denotation LFR1DILMTQSPASLSASVGETVTITCRA (SEQ ID NO: 30) LCDR1SGNIHNYLA (SEQ ID NO: 31) LFR2 WYQQKQGKSPQLLVY (SEQ ID NO: 32) LCDR2NAKTLAD (SEQ ID NO: 33) LFR3 GVPSRFSGSGSGTQYSLKINSLQPEDFGSYYC (SEQ IDNO: 34) LCDR3 QHFWSTPWT (SEQ ID NO: 35) LFR4 FGGGTKLEMKR (SEQ ID NO: 36)

>6G7 light chain DNA nucleotide Sequence (SEQ ID NO: 37)GACATTCTGATGACCCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATGAAACGC >6G7 light chain protein Sequence (SEQ ID NO: 29)DILMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEMKR >6G7 light chain protein (SEQ ID NO: 29) and DNA nucleotide (SEQ ID NO: 37)Sequence   1  D  I  L  M  T  Q  S  P  A  S  L  S  A  S  V  G  E  T  V  T 20   1 GACATTCTGATGACCCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACC  60 21  I  T  C  R  A  S  G  N  I  H  N  Y  L  A  W  Y  Q  Q  K  Q  40  61ATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAG 120  41 G  K  S  P  Q  L  L  V  Y  N  A  K  T  L  A  D  G  V  P  S  60 121GGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCA 180  61 R  F  S  G  S  G  S  G  T  Q  Y  S  L  K  I  N  S  L  Q  P  80 181AGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCT 240  81 E  D  F  G  S  Y  Y  C  Q  H  F  W  S  T  P  W  T  F  G  G 100 241GAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGTGGACGTTCGGTGGA 300 101 G  T  K  L  E  M  K  R 108 301 GGCACCAAGCTGGAAATGAAACGC 324

Example 6

Other exemplary embodiments include:

1. A binding molecule for αSyn, comprising:

one or more complementarity determining regions (CDRs) recognizingepitopes on aggregated αSyn.

2. The binding molecule of claim 1, wherein said binding molecule is anantibody or a fragment, variant, or derivative thereof.3. The antibody of claim 2, wherein said antibody is an IgG or IgM.4. The antibody fragment of claim 2, wherein said antibody fragment,variant, or derivative is a Fab, F(ab′)₂, monospecific Fab₂, bispecificFab₂, trispecific Fab₃, monovalent IgG, scFv, bispecific diabody,trispecific triabody, scFv-Fc, or minibody.5. The antibody of claim 2, wherein said antibody is humanized.murinized, and/or chimeric.6. The binding molecule of claim 1, wherein said one or more CDRs have ahigh specificity for aggregated αSyn.7. The binding molecule of claim 1, wherein said one or more CDRs have alow specificity for monomeric αSyn.8. The binding molecule of claim 1, wherein said one or more CDRs do notrecognize epitopes on monomeric αSyn.9. The binding molecule of claim 1, wherein said one or more CDRs are atleast 90% identical to the CDRs of 3A8 and/or 6G7.10. The binding molecule of claim 1, wherein said one or more CDRs areat least 95% identical to the CDRs of 3A8 and/or 6G7.11. The binding molecule of claim 1, wherein said one or more CDRscomprise of the CDRs of 3A8 and/or 6G7.12. The binding molecule of claims 9-11, wherein said one or more CDRscomprise of 3A8 heavy chain CDRs as defined by SEQ ID NOs: 4, 6, and/or8.13. The binding molecule of claims 9-11, wherein said one or more CDRscomprise of 3A8 light chain CDRs as defined by SEQ ID NOs: 13, 15,and/or 17.14. The binding molecule of claims 9-11, wherein said one or more CDRscomprise of 6G7 heavy chain CDRs as defined by SEQ ID NOs: 22, 25,and/or 27.15. The binding molecule of claims 9-11, wherein said one or more CDRscomprise of 6G7 light chain CDRs as defined by SEQ ID NOs: 31, 33,and/or 35.16. A polynucleotide encoding the polypeptide of any one of claims 1-15.17. An expression vector, comprising:

one or more polynucleotides of claim 16; and

a promoter, wherein said one or more polynucleotides is operantly linkedto said promoter.

18. The expression vector of claim 17, wherein said promoter is aneukaryote promoter.19. A bacterial host cell transformed with the expression vector ofclaim 18.20. The expression vector of claim 17, wherein said promoter is aheterologous promoter.21. The expression vector of claim 17, wherein said heterologouspromoter is a cytomegalovirus, simian virus 40, or retroviral promoter.22. The expression vector of claim 17, further comprising an encodedsignal peptide.23. The expression vector of claim 17, wherein said expression vector isa plasmid, phage, virus, or retrovirus.24. A host cell, comprising an expression vector of any one of claims20-23.25. The host cell of claim 24, wherein said host cell is mammalian.26. A method for preparing an anti-human alpha-synuclein antibody orhuman alpha-synuclein-binding fragment, derivative, or variant thereof,the method comprising:

culturing the host cell of any one of claims 19, 24-26 in a cellculture; and

isolating the anti-human alpha-synuclein antibody or humanalpha-synuclein-binding fragment, derivative, or variant thereof fromthe cell culture.

27. The method of claim 26, further comprising formulating theanti-human alpha-synuclein antibody or human alpha-synuclein-bindingfragment, derivative, or variant thereof into a sterile pharmaceuticalcomposition suitable for administration to a human subject.28. The method of claim 27, wherein the pharmaceutical composition issuitable for intravenous or subcutaneous administration.29. The method of claim 27 wherein said sterile pharmaceuticalcomposition is loaded into a nanoparticle.30. The method of claim 29, wherein said nanoparticle comprisespolyanhydride.31. A sterile pharmaceutical composition, comprising:

an anti-human alpha-synuclein antibody or human alpha-synuclein-bindingfragment, derivative, or variant thereof.

32. The sterile pharmaceutical composition of claim 31, furthercomprising:

a nanoparticle, wherein said sterile pharmaceutical composition isloaded into the nanoparticle.

33. The nanoparticle of claim 32, wherein said nanoparticle comprisespolyanhydride.34. A method of screening for the presence of aggregate alpha-synucleinin a subject, comprising:

obtaining said subject's sample; and

detecting whether aggregates of alpha-synuclein is present is saidsample by contacting the sample with a reporter and detecting bindingbetween said aggregates of alpha-synuclein and the reporter.

35. The method of claim 34, wherein the reporter comprises:

a binding molecule; and

an antibody,

wherein said binding molecule is an anti-alpha-synuclein antibody orfragment, derivative, or variant thereof; and

wherein said antibody is conjugated with an enzyme and binds to saidfirst antibody.

36. The method of claim 35, wherein said binding molecule comprises apeptide of any one of claims 1-15.37. A method of screening for the presence of aggregate alpha-synucleinin a subject, comprising:

obtaining said subject's sample;

fixing aggregate alpha-synuclein to a substrate;

adding said sample to said fixed substrate, wherein said sample isfreely suspended in solution;

adding a binding molecule, wherein said binding molecule is ananti-alpha-synuclein antibody or fragment, derivative, or variantthereof;

allowing sufficient time for said binding molecule to bind to aggregatealpha-synuclein fixed to the substrate and/or in the sample;

separating sample and any bound binding molecule from fixed aggregatealpha-synuclein;

adding a reporter, wherein said reporter is conjugated with an enzymeand may bind to said binding molecule; and

detecting whether aggregates of alpha-synuclein is present is saidsample by contacting the sample with a reporter and detecting bindingbetween said aggregates of alpha-synuclein and the reporter.

38. The method of claim 37, wherein said binding molecule comprises apeptide of any one of claims 1-15.39. A kit for assaying a cell for dopamine production, comparing:

a binding molecule which will capture aggregate alpha-synuclein in asample;

a set of reagents; and

instructions for use.

40. The kit of claim 39, wherein said binding molecule is a polypeptideof any one of claims 1-15.41. The kit of claim 39, further comprising an antibody, wherein saidantibody binds to the binding molecule and is conjugated with an enzyme.42. A system for detecting a dopamine producing cell, comprising:

a sample;

a kit comprising binding molecule which may capture aggregatedalpha-synuclein; and

a devise to detect the capture of said aggregated alpha-synuclein.

43. The system of claim 42, wherein said binding molecule is apolypeptide of any one of claims 1-15.44. The system of claim 42, further comprising an antibody, wherein saidantibody binds to the binding molecule and is conjugated with an enzyme.

What is claimed is:
 1. An antibody, or fragment, variant, or derivativethereof for aggregate αSyn, comprising: a complementarity determiningregion recognizing an epitope on aggregated αSyn and lacks crossreactivity to monomeric αSyn; and a carrier, suitable for treatingParkinson's Disease and other Lewy body- and Lewy neurite-based diseasesand for forming a complex with aggregate αSyn.
 2. The antibody, orfragment, variant, or derivative thereof of claim 1 is an IgG or IgMantibody.
 3. The antibody, fragment, variant, or derivative thereof 1,is an antigen binding fragment, a prime antigen binding fragment,monospecific divalent antigen binding fragment, bispecific divalentantigen binding fragment, trispecific trivalent antigen bindingfragment, monovalent IgG, single-chain variable fragment, bispecificdiabody, trispecific triabody, single-chain variable fragment fused to aheterologous fragment crystallizable region, or minibody.
 4. Theantibody, or fragment, variant, or derivative thereof of claim 1, ismodified to be humanized, murinized, and/or chimeric.
 5. The antibody,or fragment, variant, or derivative thereof of claim 1, conjugated to alabel.
 6. The antibody, or fragment, variant, or derivative thereof ofclaim 5, wherein the label is an enzymes, radioisotope, colloidal metal,fluorescent compound, chemiluminescent compound, or bioluminescentcompound.
 7. The antibody, or fragment, variant, or derivative thereofof claim 1, wherein said complementary determining region is at least90% identical to SEQ ID NOs: 2, 11, 20, or
 29. 8. The antibody, orfragment, variant, or derivative thereof of claim 1, having a heavychain complementary determining region as defined by SEQ ID NOs: 4, 6,or 8, and a light chain complementary determining region as defined bySEQ ID NOs: 13, 15, or
 17. 9. The antibody, or fragment, variant, orderivative thereof of claim 1, having a heavy chain complementarydetermining region as defined by SEQ ID NOs: 22, 25, or 27, and a lightchain complementary determining region as defined by SEQ ID NOs: 31, 33,or
 35. 10. A method for detecting aggregated αSyn in a sample for anearly diagnostic of Parkinson's Disease and other Lewy body- and Lewyneurite-based diseases, comprising: obtaining a sample; contacting thesample with an antibody, or fragment, variant, or derivative thereof foraggregate αSyn and lacks cross reactivity to monomeric αSyn underconditions that allow the formation of a complex between the antibody,or fragment, variant, or derivative thereof and aggregate αSyn; anddetecting the formation of the complex.
 11. The method of claim 10,wherein the antibody, or fragment, variant, or derivative thereof isconjugated to a label allowing for direct detection of the αSyn anantibody, or fragment, variant, or derivative thereof and aggregate αSyncomplex.
 12. The method of claim 11, wherein the label is an enzymes,radioisotope, colloidal metal, fluorescent compound, chemiluminescentcompound, or bioluminescent compound.
 13. The method of claim 10,further comprising: adding a second label conjugated antibody capable offorming a second complex with said αSyn antibody, or fragment, variant,or derivative thereof under conditions that allow the formation of asecond complex allowing for indirect detection of the αSyn an antibody,or fragment, variant, or derivative thereof and aggregate αSyn complex.14. The method of claim 13, wherein the label is an enzymes,radioisotope, colloidal metal, fluorescent compound, chemiluminescentcompound, or bioluminescent compound.
 15. The method of claim 10,wherein the sample is neural tissue, blood, plasma, cerebral spinalfluid, or urine.
 16. A method of treating Parkinson's Disease and otherLewy body- and Lewy neurite-based diseases, comprising: administering toa subject in need thereof an antibody, or fragment, variant, orderivative thereof for aggregate αSyn and lacks cross reactivity tomonomeric αSyn; and a carrier.
 17. The method of claim 16, wherein thedisease is Parkinson's Disease.
 18. The method of claim 16, wherein thecarrier is a nanoparticle.
 19. The method of claim 16, wherein theantibody, or fragment, variant, or derivative thereof is a single-chainvariable fragment having a heavy chain complementary determining regionas defined by SEQ ID NOs: 4, 6, or 8, and a light chain complementarydetermining region as defined by SEQ ID NOs: 13, 15, or
 17. 20. Themethod of claim 16, wherein the antibody, or fragment, variant, orderivative thereof is a single-chain variable fragment having a heavychain complementary determining region as defined by SEQ ID NOs: 22, 25,or 27, and a light chain complementary determining region as defined bySEQ ID NOs: 31, 33, or 35.